Synthesis, structure and conformation of partially-modified retro- and retro-inverso ψ[NHCH(CF3)]Gly peptides

Article abstract of DOI:10.1002/chem.200304881

Partially modified retro-(PMR) and retro-inverso (PMRI) ψ[NHCH(CF ₃)]Gly peptides, a conceptually new class of peptidomimetics, have been synthesized in wide structural diversity and variable length by aza-Michael reaction of enantiomerically p

Full text of DOI:10.1002/chem.200304881

FULL PAPER
Synthesis, Structure and Conformation of Partially-Modified Retro- and
Retro-Inverso y[NHCH(CF3)]Gly Peptides
Alessandro Volonterio,*[b] Stefano Bellosta,[c] Fabio Bravin,[b] Maria Cristina Bellucci,[d]
Luca Bruche¬,[b] Giorgio Colombo,[e] Luciana Malpezzi,[b] Stefania Mazzini,[d]
Stefano V. Meille,[b] Massimiliano Meli,[e] Carmen RamÌrez de Arellano,[f] and
Matteo Zanda*[a]
Abstract: Partially modified retro-
(PMR) and retro-inverso (PMRI)
y[NHCH(CF3)]Gly peptides, a concep-
tually new class of peptidomimetics,
have been synthesized in wide structural
diversity and variable length by aza-
Michael reaction of enantiomerically
pure a-amino esters and peptides with
enantiomerically and geometrically pure
N-4,4,4-trifluorocrotonoyl-oxazolidin-2-
ones. The factors underlying the ob-
served moderate to good diastereocon-
trol have been investigated. The con-
peptides, was evidenced, as a likely
consequence of two main factors: 1) se-
vere torsional restrictions about sp3
bonds in the [CO-CH2-CH(CF3)-NH-
CH(R)-CO] module, which is biased by
the stereoelectronically demanding CF3
group and the R side chain; 2) formation
of nine-membered intramolecularly hy-
drogen-bonded rings, which have been
clearly detected both in CHCl3 solution
and in some crystal structures. The
former factor seems to be more impor-
tant, as turn-like conformations were
found in the solid-state even in the
absence of intramolecular hydrogen
bonding. The relative configuration of
the -C*H(CF3)NHC*H(R)- stereogenic
centers has a major effect on the stability
of the turn-like conformation, which
seems to require a syn stereochemistry.
X-ray diffraction and ab initio computa-
tional studies showed that the [-
CH(CF3)NH-] group can be seen as a
sort of hybrid between a peptide bond
mimicand a proteolytictransition state
analogue, as it combines some of the
properties of a peptidyl -CONH- group
(low NH basicity, CH(CF3)-NH-CH
À
backbone angle close to 1208, C CF3
formations
of
model
PMR-
bond substantially isopolar with the
ˆ
y[NHCH(CF3)]Gly tripeptides have
been studied in solution by 1H NMR
spectroscopy supported by MD calcula-
tions, as well as in the solid-state by
X-ray diffraction. Remarkable stability
of turn-like conformations, comparable
to that of parent malonyl-based retro-
C O) with some others of the tetrahe-
dral intermediate [-C(OX)(OÀ)NH-] in-
volved in the protease-mediated hydrol-
ysis reaction of a peptide bond (high
electron density on the CF3 group,
tetrahedral backbone carbon).
Keywords: conformation analysis ¥
fluorine
¥
Michael addition
¥
peptidomimetics
quence of fast in vivo degradation by proteolytic enzymes,
represents a serious pharmacological drawback.[1] The need of
drug-like molecules retaining both activity and potency of the
parent peptides, while at the same time much more stable and
Introduction
Peptides are essential to virtually every biochemical process
but their low bioavailability, which is principally a conse-
[a] Dr. M. Zanda
[d] Dr. M. C. Bellucci, Dr. S. Mazzini
C.N.R.–Istituto di Chimica del Riconoscimento Molecolare (ICRM)
Sezione ™A. Quilico∫, via Mancinelli 7
20131 Milano (Italy)
Dipartimento di Scienze Molecolari Agroalimentari
¡
Universita degli Studi di Milano
Via Celoria 2, 20133 Milano (Italy)
Fax : (‡39) 02 23993080
[e] Dr. G. Colombo, M. Meli
E-mail: matteo.zanda@polimi.it
C.N.R.–ICRM, Sezione Milano-1
via M. Bianco 9, 20131 Milano (Italy)
¬
[b] Dr. A. Volonterio, F. Bravin, Prof. Dr. L. Bruche, Dr. L. Malpezzi,
Prof. Dr. S. V. Meille
Dipartimento di Chimica, Materiali ed Ingegneria Chimica ™G. Natta∫
Politecnico di Milano
[f] Prof. Dr. C. RamÌrez de Arellano
¬
Departamento de QuÌmica Organica
Facultad de Farmacia, Universidad de Valencia
46100 Burjassot, Valencia (Spain)
via Mancinelli 7, 20131 Milano (Italy)
[c] Dr. S. Bellosta
Supporting information for this article is available on the WWW under
http://www.chemeurj.org/ or from the author.
Dipartimento di Scienze Farmacologiche
¡
Universita degli Studi di Milano
via Balzaretti 9, 20133 Milano (Italy)
4510
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/chem.200304881
Chem. Eur. J. 2003, 9, 4510 4522

4510 4522
orally active, has been a major driving-force for the develop-
ment of a variety of peptide mimics. Two very attractive ways
of generating bio-active peptide mimics with improved
biostability are: a) to replace a peptide bond with a surrogate
unit X, which is usually symbolized as y(X);[2] b) to reverse all
or some of the peptide bonds (NH-CO instead of CO-NH)
giving rise to the so called retro- or partially-modified-retro
(PMR) peptides, respectively.[3, 4] When the stereochemistry
of one or more amino acids of the reversed segment is
inverted, the resulting pseudo-peptide is termed as retro-
inverso. A malonic unit is classically incorporated to provide
partially-modified retro-peptides, while the direction can be
restored incorporating an additional gem-diaminoalkyl unit.
Recently, we communicated a new strategy for generating
peptidomimeticsequences, based on the idea of combining
both the ™surrogate-unit∫ and the ™direction-reversal∫ strat-
Further important biological and physicochemical features
are expected to arise from the presence of the [CH(CF3)NH]
group. For example, the y[NHCH(CF3)] surrogate should be
very stable toward proteolyticdegradation. Furthermore, the
stereoelectronically demanding CF3 group is expected to
constraint the molecule; this limits the number of accessible
conformational states and lead to peptidomimetics displaying
well-defined conformational motifs. Last but not least, it
should modify the binding properties of the parent peptide,
changing the hydrogen bonding or coordinative features of
the ligand in the putative receptor sites.[8]
We now report in full on: 1) the solution-phase chemistry
which leads to PMR and PMRI y[NHCH(CF3)]Gly peptides
(R ˆ H in Figure 1), 2) the results of NMR, molecular
modelling and X-ray studies on their propensity to assume
turn-like conformations in solution and in the solid state, and
3) the structural properties of the [NHCH(CF3)] module
using X-ray diffraction and ab initio calculations to character-
ize its molecular electrostatic potential surface.
egies in
a
novel class of pseudo-peptides with
a
y[NHCH(CF3)] instead of the y(NHCO) unit featured by
retro-peptides (Figure 1).[5]
Results and Discussion
Chemistry: We envisaged a syntheticapproahc to the
y[NHCH(CF3)]-containing dipeptide units involving aza-
Michael reaction of a series of l-a-amino esters 1 (Scheme 1)
with enantiomerically and geometrically pure Michael accept-
ors (S,E)-2, prepared according to literature methods.[9]
Figure 1. Conventional retro- and retroinverso peptides, and
y[NHCH(CF3)] retropeptides.
A first intuitive consequence of this structural modification
is that the symmetry of conventional malonyl PMR peptides is
broken, resulting in the generation of two families of
regioisomericPMR- y[NHCH(CF3)] and -y[CH(CF3)NH]
peptides (Figure 2),[6] each one existing in two epimeric forms
at the CF3-substituted carbon. In this way a great diversity of
structurally related, potentially bioactive molecules can be
created.[7]
Scheme 1. Aza-Michael reaction with amino-ester nucleophiles 1.
This reaction is extraordinarily simple and efficient, if one
considers that examples of aza-Michael reactions involving
chiral a-amino esters and 4-substituted Michael acceptors are
very scarce in the literature.[10] Pseudo-dipeptides 3 were
formed in excellent yields simply by mixing the hydrochlor-
ides, or PTSA salts, 1 (2 equiv) with (S,E)-2 and sym-collidine
(TMP) (4 equiv) in the appropriate solvent, and stirring at
room temperature for 16 88 hours (Table 1).[11] The solvent
has a strong influence on stereoselectivity, as shown by
experiments carried out on the model reaction between l-1a
and 2a. The best results were obtained in CH2Cl2 (entry 1),
while a substantial drop of de was observed with more polar
solvents such as ethanol, acetonitrile, THF, DMF or mixtures
(entries 2 5). The base also plays an active role, as
demonstrated by the fact that the use of DABCO (both in
CH2Cl2 and toluene), instead of TMP, accelerates the reaction
of 1b with 2a but lowers the de of the product 3c (entries 9
and 10) in comparison with TMP (entry 8). In the absence of a
base (entry 11), namely pre-generating the free a-amino ester
Figure 2. Different
isomers
of
y[NHCH(CF3)]Gly-
and
y[CH(CF3)NH]Gly retropeptides.
Chem. Eur. J. 2003, 9, 4510 4522
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. Volonterio, M. Zanda et al.
FULL PAPER
from the hydrochloride by treatment with NaHCO3, the
diastereoselectivity was similar to that achieved with TMP. In
the light of these results, CH2Cl2 and TMP were used as,
respectively, the solvent and the base of choice for the
preparation of pseudo-dipeptides 3a l. The facial diastereo-
selectivity of these reactions is mainly controlled by the
nucleophiles 1. In fact, in all cases l-configurated 1 (Scheme 1
and Table 1, entries 1 20) attack preferentially the Si face of
(S)-2a producing new (S)-centers, whereas enantiomeric d-
Ala-OMe 1a (entry 23) attacks the Re face producing the (R)-
configurated diastereomer 5a, although with lower diaster-
eocontrol (mismatch).[12] Moreover, compound 3e was pro-
duced with good diastereocontrol when l-Val-OBn 1b was
reacted with the achiral Michael-acceptor 2c (entry 14), while
achiral H-Gly-OEt 1g (entry 21) and Bn-Gly-OEt 1h (en-
try 22) added to (S)-2a without stereocontrol, affording
equimolar mixtures of 3k and 3l, respectively. It is therefore
apparent that the stereoelectronic features of the a-amino
ester R1 side chain have a major impact on the degree of
diastereoselectivity, which follows the trend iPr > iBu > Me
> Bn > H (de up to 78% in the case of 3d, entry 13). Modest
de was obtained with the cyclic a-amino ester l-Pro-OBn 1 f
(entry 20), but the reaction occurred also in this case with very
good yield. On the other hand, the R3 substituent on the
oxazolidinone stereocenter has a lower effect on the stereo-
selectivity. In fact, (S)-configurated products 3d (R3 ˆ iPr,
entry 13), 3c (R3 ˆ Bn, entry 11) and 3e (R3 ˆ H, entry 14)
were obtained from l-Val-OBn 1b in 78, 72 and 65% de,
respectively, with little variation and the same sense of facial
selectivity. No meaningful effect is exerted by the X group
of the nucleophile, as shown by the fact that l-Phe-OBn
(1 f, entry 15) and l-Phe-OtBu (1g, entry 16) added to 2a
with nearly identical diastereocontrol (43 and 45%, respec-
tively).
The results described above can be rationalized if one
considers that in the absence of chelating agents N-(E)-enoyl-
oxazolidin-2-ones 2 exist in transoid conformation (as por-
trayed throughout the paper),[13] with the R3 substituent
ˆ
pointed away from the C C bond, thus exerting little control
of the facial selectivity. In contrast, the R1 side chain of a-
amino esters 1 should be spatially close to the forming
stereogenicecnter in the transition state, therefore its
influence is much more important. Chelation with Lewis
acids for biasing the oxazolidin-2-ones 2 in cisoid conforma-
tion was tried with little success. For example, treatment of 2a
with Sc(OTf)3 (entry 12),[14] followed by addition ofL À 1c
and TMP, produced 3d in 58% de and ꢀ85% yield
(determined by 19F NMR of the crude reaction mixture) after
30 h at RT. The reaction time had no effect on diastereose-
lectivity, in fact 3h was formed from 1e and 2a with the
identical 50% de after 16 h (ca. 50% conversion, entry 18) or
after 88 hours as well (entry 17), as shown by NMR of the
crude reaction mixture. This strongly supports the conclusion
that adducts 3 are formed irreversibly, and the stereochemical
outcome is under kinetic control.
It is worth noting that stereocontrol is totally absent in the
aza-Michael reaction of l-Ala-OMe 1a with methyl 4,4,4-
trifluorocrotonate, which was chosen initially as the Michael
acceptor, and the reaction is much slower (one month, RT).
This is likely to be a consequence of the lower conformational
rigidity and electrophilicity of 4,4,4-trifluorocrotonate with
respect to the oxazolidinone acceptors 2.
Table 1. Aza-Michael reaction with amino-ester nucleophiles 1.
Entry Major product AA Acceptor Oxazolidinones R1
R2
R3
X
Solvent
Base
t [h] Yield [%][c] de [%][d]
1
2
3
4
5
6
7
8
9
10
11
12[e]
13
14
15[a]
16
17[a]
18
19
20
21
22
23
3a
3a
3a
3a
3a
3b
3c
3c
3c
3c
3c
3c
3d
3e
3 f
3g
3h
3h
3i
1a
1a
1a
1a
1a
1a
1b
1b
1b
1b
1b
1b
1b
1b
1c
1d
1e
1e
1e
1 f
1g
1h
d-1a
2a
2a
2a
2a
2a
2b
2a
2a
2a
2a
2a
2a
2b
2c
2a
2a
2a
2a
2b
2a
2a
2a
2a
Me
Me
Me
Me
Me
Me
iPr
iPr
iPr
iPr
iPr
iPr
iPr
iPr
Bn
Bn
iBu
iBu
iBu
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Bn
Bn
Bn
Bn
Bn
Me
Me
Me
Me
Me
CH2Cl2
EtOH
MeCN
TMP
TMP
TMP
16
16
40
40
40
64
64
6.5
6.5
6.5
90
80
80
n.d.[b]
> 98
91
50
20
31
26
29
60
72
72
69
68
72
58
78
65
43
45
50
50
60
29
< 5
< 5
31
THF/DMF 4:1 TMP
PhH/DMF 4:1
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
toluene
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
TMP
TMP
TMP
TMP
DABCO
DABCO
None
TMP
TMP
TMP
TMP
TMP
TMP
TMP
TMP
TMP
TMP
TMP
TMP
iPr Me
Bn
Bn
Bn
Bn
Bn
Bn
iPr Bn
H
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
92
41[d]
58[d]
65[d]
60
> 98
60
68
68
24
60
88
16
68
40
40
72
16
85
> 98
90
Bn
Bn
78
t-Bu CH2Cl2
Bn
Bn
> 98
> 98
50[d]
> 98
> 98
> 98
70[d]
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
iPr Bn
3j
3k
3l
-(CH2)3-
H
H
Me
Bn
Bn
Bn
Bn
Bn
Et
Et
H
Bn
H
5a
Me
> 98
[a] PTSA salt of the amino acid was used. [b] Not determined. [c] Overall isolated yield of both diastereomeric products, unless otherwise stated.
1
[d] Determined by 500 MHz H and 19F NMR. [e] Oxazolidinone 2a was prior treated with Sc(OTf)3 in CH2Cl2 at RT for 30 min.
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Pseudopeptides
4510 4522
We next explored the viability of the method for preparing
longer PMR and PMRI y[NHCH(CF3)]Gly peptides by using
N-terminally unprotected peptides as nucleophiles. Satisfac-
torily, dipeptides 6a c (Scheme 2) reacted with 2a affording
in excellent yields and good stereocontrol the tripeptide
Scheme 2. Aza-Michael reaction with dipeptide nucleophiles 6.
mimics 7a c (Table 2). It is worth noting that while, in
analogy with simple a-amino esters 1, H-Val-Ala-OMe 6b
(entry 2) added more stereoselectively (70% de) than H-Phe-
Ala-OMe 6a (46% de, entry 1), we observed an unexpectedly
strong effect by the remote stereocenter of the second amino-
acid residue. In fact, the dipeptide H-Val-d-Ala-OMe 6c
(entry 3) scored a much lower diastereocontrol than 6b,
affording 7c in 54% de.
Scheme 3. i) LiOH, H2O2; ii) HATU/HOAt, sym-collidine, DMF, a-amino
ester.
Table 2. Aza-Michael reaction with dipeptide nucleophiles 6.
Entry
Product
Dipeptide
R1
R2
Yield [%][a]
de [%][b]
Bio-assays: Within the frame of an ongoing program aimed at
the identification of fluorinated peptide mimics for inhibition
of matrix metalloproteinases (MMPs), the three model
compounds 9b,c,e were randomly chosen to study their effect
on MMP-9 (gelatinase B) expression and activity in human
macrophages. In contrast with PMR y[NHCH(CF3)]Gly-
peptidyl hydroxamates,[7c] some of which showed moderate
activity, the three assayed molecules showed negligible
activity, confirming the well known importance of the
terminal hydroxamate function for MMPs inhibition.
1
2
3
7a
7b
7c
6a
6b
6c
Bn
iPr
iPr
l-Me
l-Me
d-Me
> 98
90
> 98
46
70
54
[a] Overall isolated yield of both diastereomericproducts. [b] Determined
by 500 MHz H and 19F NMR.
1
With a number of PMR y[NHCH(CF3)]Gly peptides at
hand we addressed the chemoselective cleavage of the
oxazolidinone auxiliary. This result was achieved in 55
82% yields upon treatment of the oxazolidinone pseudopep-
tides 3 7 (Scheme 3) with LiOH/H2O2 (30 min, 08C).[15] The
resulting pseudo-di- and tri-peptides with a terminal CO2H
group were purified by FC, then coupled with another a-
amino ester (HATU/HOAt, sym-collidine, DMF). The final
PMR and PMRI tripeptides 9a e and tetrapeptide 10,
orthogonally protected at the carboxy end groups (except
9e) and therefore suitable for further selective elongation,
were obtained in quantitative yields, often as solid materi-
als.[16]
Secondary structure of PMR y[NHCH(CF3)]Gly peptides: b-
Turns are one of the three major structural elements of
peptides and proteins. A b-turn is characterized by the
presence of a ten-membered intramolecularly hydrogen-
ˆ
À
bonded (C O ¥¥¥ H N) ring involving four amino-acid resi-
dues (from i through the i‡3 positions).[18] It has been shown
A very interesting feature of PMR y[NHCH(CF3)]Gly
peptides 3 10 is their weakly basicnature, which is due to the
presence of the strongly electron-withdrawing CF3 in the a-
position to the amino group. As a matter of fact, PMR
y[NHCH(CF3)]Gly peptides 3 10 did not form stable salts
with trifluoroacetic acid or 2n hydrochloric acid, as assessed
1
by H and 19F NMR spectroscopy.[17] In terms of basicity, the
[NHCH(CF3)] moiety resembles a retropeptide unit [NHCO]
more closely than a conventional secondary amine moiety.
that very simple PMR-peptides, such as the triamide 11
(Figure 3), adopt turn-like nine-membered folding patterns
Chem. Eur. J. 2003, 9, 4510 4522
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A. Volonterio, M. Zanda et al.
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Figure 3. Conformations of Gellman×s and y[NHCH(CF3)]Gly peptides.
*
Figure 4. Amide proton (NHb) NMR chemical shifts of 12 ( , c ˆ 3.0mm;
~
&
^
, c ˆ 0.05mm), 13 ( , c ˆ 3.0mm) and 14 ( , c ˆ 3.0mm) in CDCl3 solutions
À
ˆ
as a function of temperature.
with an intramolecular N H ¥¥¥ O C hydrogen bond in the
solid state, as well as in low-polarity solvent solutions.[19]
As a starting point of our investigation on the properties of
PMR y[NHCH(CF3)]Gly peptides, we decided to assess
whether some conformational analogies exist with the un-
fluorinated parent molecules. Racemic y[NHCH(CF3)]-dia-
mide 12 (Figure 3) was synthesized in good overall yield by
aza-Michael of H-Gly-OBn (1i) with the achiral acceptor 2c
(Scheme 4), followed by a standard reaction sequence per-
formed on the adduct 3m.
lecular hydrogen bonds are involved in this range of temper-
atures. In contrast, at lower temperatures (from 263 to 213 K)
the NHb chemical shift did not change significantly with
dilution (Figure 4). A very high Dd/DT 0.0111 was also
observed at this concentration. The data above can be
interpreted in terms of formation of a more stable intra-
molecularly hydrogen-bonded folded structure of 12 at low
temperature (see Figure 3), in analogy with Gellman×s
unfluorinated PMR-peptide 11.
However, 2D ROESY experiments performed on 12 at
both 213 and 293 K did not show NOE interactions that could
unambiguously confirm the presence of a folded conforma-
tion, because also the distance values of the extended
structure are within 5 ä.
Next, in order to investigate the effect of the configuration
of the CF3-substituted stereogenic center on the conformation
of PMR y[NHCH(CF3)]Gly peptides in solution, we selected
the two diastereomeric O-Bn PMR y[NHCH(CF3)]Gly-
peptidyl hydroxamates 13 and 14.[21]
Scheme 4. Synthesis of the model y[NHCH(CF3)]-diamide 12.
In a weakly hydrogen bonding solvent, such as CDCl3, both
the absolute value and the temperature dependence of an
amide proton chemical shift provide important structural
information. To assess whether the amide NHb proton of 12 is
involved in hydrogen bonding, we first investigated the
™reduced temperature coefficient∫ Dd/DT.[20] 1H NMR vari-
able temperature studies from 293 to 213 K, performed on a
3mm solution of 12 in CDCl3, showed that the chemical shifts
of the amide protons NHb moved downfield of 0.81 ppm, with
a Dd/DT 0.0101 (see Figure 4). This very high Dd/DT ratio
strongly suggests that the NHb proton forms hydrogen bonds
of increasing stability as the temperature is lowered.
In order to determine if such chemical shift variations were
due to the formation of intra- or intermolecular hydrogen
bonds, another series of 1H NMR experiments was performed
at 0.05mm of 12 in CDCl3, and the spectra were acquired at
different temperatures ranging from 293 to 213 K. In the
range between 293 and 273 K the NHb chemical shift of 12
moved upfield of 0.1 ppm with the dilution in comparison with
the 3mm solution (see Figure 4), indicating a partial disrup-
tion of hydrogen bonds. This strongly suggests that intermo-
In fact, we have recently shown via X-ray diffraction that in
the solid state 13 displays a hydrogen-bonded turn-like
conformation (Figure 5) closely resembling that of unfluori-
nated PMR peptides.[19] 1H NMR variable temperature
experiments (from 293 to 213 K), performed on 3mm
solutions of 13 and 14 in CDCl3 showed, respectively, a
0.99 ppm (Dd/DT 0.0124) and 0.26 ppm (Dd/DT 0.00325)
deshielding for the NHb protons (see Figure 4).[22] It is also
immediately apparent that 13 exhibits NHb chemical shifts
downfield at all temperatures with respect to 14, which in turn
was always deshielded with respect to 12. In solution, in a
regime of fast exchange, the observed chemical shifts are
averages of the shifts of the non-hydrogen bonded and
hydrogen bonded species, so it is possible qualitatively to
conclude that: 1) the NHb protons of both 13 and 14 are more
involved in hydrogen bonding than the NHb of 12; 2) the NHb
protons of both 13 and 14 form hydrogen bonds of increasing
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Pseudopeptides
4510 4522
Table 3. Intramolecular NOE interactions and interproton distances
values [ä] for 13.
Experimental NOE
Model distances
NHb ¥¥¥ H-1 (pro R)
NHb ¥¥¥ H-2 (pro S)
NHb ¥¥¥ H-3
NHb ¥¥¥ H-6
NHb ¥¥¥ H-7
NHb ¥¥¥ H-8 (pro R)
NHb ¥¥¥ H-9 (pro S)
NHb ¥¥¥ H-12
2.58
3.14
4.36
2.91
3.48
4.53
4.12
4.04
2.06
3.49
4.84
4.75
2.40
3.49
4.05
4.97
3.59
4.34
4.50
4.88
2.44
3.63
1.76
4.23
2.80
2.34
3.12
2.59
1.76
2.39
3.43
2.77
2.64
3.58
1.75
2.70
3.52
2.99
2.43
2.63
1.74
3.53
2.52
3.84
2.14
2.13
3.64
2.96
2.51
NHb ¥¥¥ NHa
NHb ¥¥¥ CH3 (pro S)
NHb ¥¥¥ CH3 (pro R)
NHb ¥¥¥ Phe(A) (pro S)
Phe(A) ¥¥¥ H-1 (pro R)
Phe(A) ¥¥¥ H-2 (pro S)
Phe(B) ¥¥¥ CH3 (pro S)
Phe(B) ¥¥¥ H-12
Phe(B) ¥¥¥ NHa
Phe(B) ¥¥¥ H-3
Phe(B) ¥¥¥ H-4 (pro R)
Phe(B) ¥¥¥ H-5 (pro S)
Phe(B) ¥¥¥ H-10 (pro R)
Phe(B) ¥¥¥ H-11 (pro S)
H-1 ¥¥¥ H-2
H-1 (pro R) ¥¥¥ H-6
H-3 ¥¥¥ H-4 (pro R)
H-3 ¥¥¥ H-5 (pro S)
H-3 ¥¥¥ H-10 (pro R)
H-3 ¥¥¥ H-11 (pro S)
H-4 ¥¥¥ H-5
H-4 (pro R) ¥¥¥ H-11 (pro S)
H-5 (pro S) ¥¥¥ H-11 (pro S)
H-4 (pro R) ¥¥¥ H-10 (pro R)
H-6 ¥¥¥ H-8 (pro R)
H-6 ¥¥¥ H-9 (pro S)
H-8 ¥¥¥ H-9
H-7 ¥¥¥ CH3 (pro R)
H-7 ¥¥¥ CH3 (pro S)
H-7 ¥¥¥ NHa
Figure 5. Crystal conformation of PMR y[NHCH(CF3)]Gly-peptidyl
hydroxamates 13.
stability as the temperature is lowered; 3) the upfield shift and
the smaller variation of chemical shift for the NHb proton of
14 are consistent with, respectively, a lower proportion of
hydrogen bonding and a lower dependence of its folding
pattern on the temperature in comparison with its diaster-
eoisomer 13. In order to assess whether such hydrogen
bonding is inter- or intramolecular, concentration-depend-
ence experiments (not shown in Figure 4) were carried out:
the NHb chemical shifts of 13 and 14 did not change with
dilution (down to 0.05 mm) at constant temperature, thus
demonstrating that the hydrogen bonds are predominantly
intramolecular.
H-7 ¥¥¥ H-12
H-6 ¥¥¥ NHa
H-10 ¥¥¥ H-11
H-8 (pro R) ¥¥¥ NHa
H-9 (pro S) ¥¥¥ NHa
H-12 ¥¥¥ NHa
H-12 ¥¥¥ CH3 (pro R)
H-12 ¥¥¥ CH3 (pro S)
NHa ¥¥¥ CH3 (pro R)
NHa ¥¥¥ CH3 (pro S)
CH3 ¥¥¥ CH3
Actually the NOE data must be considered as the average
of the interactions occurring for many conformations. In the
case of 13 2D ROESY spectra performed at 213 and 293 K,
allowed to obtain specific NOE interactions (see Figure 6 and
conformation is already present at 293 K, indicating a strong
natural tendency of this molecule to bend.
2D ROESY experiments performed under the same
experimental conditions on 14 showed some differences
concerning the NOE, in comparison with its diastereoisomer
13. In particular, we observed that the NOE contacts between
the NHb amide proton and H-6, H-7 (strong interactions) are
preserved, while the NOE between NHb and the H-3 proton
here is lacking. Weaker contacts of NHb with H-8 and H-9
with respect to 13 were also detected. Thus, one can say that
also the hydroxamate 14 shows a certain tendency to fold in
Figure 6. The arrows indicate the experimental NOEs determining the
folded structure of 13.
Table 3) that unambiguously confirmed that in addition to the
extended conformations, this compound adopts stable folded
structures in solution. In particular, NHb shows contacts with
H-6, H-7 (strong interactions), H-3, H-8 and H-9 protons
(weak interactions). It is interesting to note that the folded
ˆ
solution, but the turn-like conformation featuring a C O ¥¥¥
Hb-N intramolecular hydrogen bond here is less populated.
Chem. Eur. J. 2003, 9, 4510 4522
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4515

A. Volonterio, M. Zanda et al.
FULL PAPER
Table 4. Selected geometrical parameters for 3a, 9a, 9c and 13.
3a 9a 13 9c
This result is in accord with the minor chemical shift variation
found for the NHb (see Figure 4), which confirms that the
molar fraction of the hydrogen bonded conformations is
lower. These findings evidence that the configuration of the
CF3-substituted carbon has an important effect on the folding
of PMR y[NHCH(CF3)]Gly peptides in solution.[23]
Because of the large conformational motions expected for
the 13, MD simulations were performed without experimental
constraints, which provided a set of structures that, after
minimisation, converge in many folded structures at low
energy. The conformation reported in Figure 7 (E ˆ
136 kcalmolÀ1) features the best agreement with the exper-
imental NOE data (Table 3) and presents an nine-membered
bond lengths
À
N2 C13
1.453(3)
1.466(3)
1.522(3)
1.504(3)
1.444(3)
1.450(3)
1.519(4)
1.511(4)
1.449(3)
1.462(3)
1.522(4)
1.531(4)
1.427(6)
1.441(6)
1.536(6)
1.513(7)
À
N2 C15
À
C12 C13
À
C13 C14
bond angles
C13-N2-C15
N2-C13-C14
N2-C13-C12
C12-C13-C14
torsion angles
116.8(2)
117.0(2)
119.2(2)
119.3(4)
108.7(2)
111.9(2)
108.5(2)
108.8(2)
112.3(2)
109.8(2)
113.5(2)
112.0(2)
107.4(2)
114.4(4)
108.0(4)
109.2(4)
C14-C13-N2-C15 À 97.8(3)
À 96.7(3)
141.6(2)
À 81.9(3)
157.0(2)
À 69.1(3)
165.7(3)
113.2(2)
À 75.2(4)
163.0(2)
À 74.9(6)
À 163.3(5)
70.8(5)
À 164.2(4)
À 99.2(6)
138.7(7)
C12-C13-N2-C15 142.4(2)
C11-C12-C13-N2 À 79.0(2)
C11-C12-C13-C14 161.1(2)
C13-N2-C15-C16 À 68.3(3)
À 71.6(3)
163.1(2)
À 94.3(3)
142.5(3)
À 173.6(2)
C13-N2-C15-C17
N1-C11-C12-C13
168.5(2)
177.7(2)
119.6(4)
1.469 ä reported for secondary amines.[27] Values are rather in
3
2
À
the range expected for C(sp ) N(sp ) single bonds. In all of
the four structures the amine hydrogen atom, experimentally
located by Fourier methods, is displaced from the plane
defined by N2 and the adjacent C13 and C15 atoms, showing a
distorted sp3 geometry of N2.
The local conformation of the common part of the four
molecules is well described by the torsion angles around the
À
À
À
À
five bonds C11 C12, C12 C13, C13 N2, N2 C15 and
À
C15 C16 (see Table 4). The similarities among the four
compounds are remarkable: indeed the conformational
preferences towards trans or gauche arrangements of these
dihedral angles are rather well defined and seem to be only
marginally influenced by the presence of an intramolecular
hydrogen bond, as found in 13. Specifically, the torsion angle
Figure 7. Conformation of 13 obtained from molecular modelling.
ˆ
intramolecularly hydrogen bonded ring with a C O ¥¥¥ H-N
distance of 2.18 ä.[24] It is worth noting that the bond angle
(CF3)CH-NH-C is 118.548 predicting a widened geometry of
the amine nitrogen atom N4 that is confirmed by experimen-
tal evidence (see below). The model distances reported in
Table 3 are in good agreement with the solid state, where the
X-ray analysis of this compound showed a folded structure
with a distance of 1.95 ä for the hydrogen bond.[7c]
À
around C12 C13 is dictated by the expected preferential
location of the CF3 group, rather than N2, trans to C11.
À
Similarly, in the case of the torsion angles around N2 C15, the
™larger∫ sp3 carbon C17, rather than the sp2 C16, is arranged
trans to the C13 atom, which has a bulky environment. The
conformation around C13 N2 is more difficult to account for
just considering local features. However, all of the four
compounds display a conformation with the C12 nearly trans
to C15, possibly due to cooperative conformational effects.
À
Structural properties of the y[NHCH(CF3)] unit: A study on
the structural and geometric properties of the
y[NHCH(CF3)] group was carried out by means of X-ray
diffraction and computational methods, also with the aim of
understanding the role of such units in promoting turn-like
structures in PMR y[NHCH(CF3)]Gly peptides.
Analysis of the X-ray structures of 3a, 9a, 9c (Figure 8)[25]
and of the known hydroxamate 13 (Figure 5)[7c] highlighted
the distortion of the sp3 nitrogen atom N2: the C13-N2-C15
angle is in fact close to 1208 (see Table 4). This value is
comparable with that found in amides and somewhat larger
than the already wide average of 116.658 determined for 203
secondary amine structures having the appropriate structural
features found in a Cambridge Structural Database search.[26]
À
Finally, the conformation around C11 C12 appears to favor a
transoid arrangement (i.e., from trans to skew) of N1 with
respect to C13, depending upon the nature of the groups
bound to N1. The basicfeatures of the turn-like structure are
in essence related to the gauche, trans, gauche sequence of,
respectively, the torsion angles C11-C12-C13-N2, C12-C13-
N2-C15 and C13-N2-C15-C16, with gauche angles of the same
sign. As readily observable in Figure 8, this property is
common to all of the molecules discussed herein, with the
notable exception of 9c. In all of the four crystal structures,
the local conformations discussed above seem to bring the
stereoelectronically demanding CF3 group in a stereochemi-
cally favorable environment. In the case of 3a, 9a and 13 this
position is represented by the exterior surface of the turn: it is
in this respect clear that the relative configuration of the CF3-
À
À
The N2 C13 and N2 C15 bond lengths are very similar in 3a,
9a and 9c and invariably shorter than the average value of
4516
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 4510 4522

Pseudopeptides
4510 4522
À
C13 CF3 group behaves as a
valid mimicof the retro-amide
carbonyl group in Gellman×s
retropeptides.
In order to verify the respec-
tive influences of the neighbor-
ing CF3 and of the substituents
directly bonded to the NH
group, a series of ab initio
geometry optimization calcula-
tions was performed. For the
sake of comparison, analogous
calculations were carried out on
the structure TI (Figure 9),
which represents a mimic of
the so called ™tetrahedral inter-
mediate∫ in the protease medi-
ated hydrolysis reaction, on
model y[NHCH(CF3)]-amides
15a
c
(Figure 9), and on
y[NHCH(CFnH3Àn)] structures
(n ˆ 0 2) 16 18, which have
lower degrees of fluorination
(Figure 10). These calculations
were accomplished in order to
check and compare structural
and electronic features of the
tetrahedral intermediate TI and
those of the fluorinated pepti-
domimetics 15 18. Several
studies have demonstrated that
the ™tetrahedral intermediate∫,
closely resembling (according
to Hammond×s postulate) the
transition state leading to the
hydrolysis of amides or esters, is
characterized by the presence
of a negatively charged oxygen
atom bound to an sp3 carbon,
belonging to the scissile amide
or ester group.[28] Other studies
on the mechanism of serine
proteases in different condi-
tions[29] have shown that the
tetrahedral intermediate is a
Figure 8. ORTEP drawings of 3a, 9a and 9c.
bearing C13 atom and the C15 amino-acid center, which is
identical in 3a, 9a and 13 and opposite in 9c, has a key role in
determining the stability of the turn-like conformation,
whereas the absolute configurations of C13 and C15 fix the
À
sign of the torsion angles around the adjacent C13 N2 and
À
C13 C12 bonds, and thus the chirality of the turn. These
findings are in agreement with the results obtained by
1H NMR spectroscopy in solution on the hydroxamates 13
and 14, and strongly suggest that a syn configuration of the [-
CH(CF3)NHCH(R)-] fragment is a fundamental requisite for
the formation of a stable turn-like conformation. One can
therefore say that, with the appropriate configuration, the
Figure 9. Calculated structures of model PMR y[NHCH(CF3)]Gly frame-
works and tetrahedral hydrolysis intermediate TI.
Chem. Eur. J. 2003, 9, 4510 4522
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4517

A. Volonterio, M. Zanda et al.
FULL PAPER
À
Table 5. CA-NF-X bond angle and CA NF bond length values for the
optimized peptide structures.[a]
À
Compound
CA-NF-X
angle [8]
CA-NF-HF1
angle [8]
CA NF bond
length [ä]
Figure 10. Calculated structures of model PMR y[NHCH(RF)]Gly frame-
works.
15a (X ˆ HF2
)
112.625
120.791
120.526
118.679
119.896
120.791
117.422
112.877
109.501
111.057
108.300
108.624
109.501
110.330
1.44
1.45
1.45
1.47
1.45
1.45
1.49
15b (X ˆ CB)
15c (X ˆ CB)
16 (X ˆ CB)
17 (X ˆ CB)
18 (X ˆ CB)
TI
good model to explain reactivity and draw mechanistic
conclusions.
Simplified models catching all the basic chemical and
conformational features of the y[NHCH(CFnH3Àn)] systems
and of TI were used in order to reduce the computation time
and allow the use of a suitable level of theory in the QM ab
initio calculations. Ab initio calculation were performed in the
perspective of calculating and comparing the stereoelectronic
features of our model compounds to those of TI, which is
impossible to isolate and whose properties are not easily
measurable. Moreover, using a high level of theory, trends in
the related compounds examined can be evidenced with high
accuracy.
The starting structure of each model peptide was built using
the Macromodel package with a spatial disposition of the
groups mimicking their respective disposition in the X-ray
structure (see Supporting Information). Each structure was
first minimized through a Molecular Mechanics approach
using the Amber[30] force field. As expected, the bond angle of
the CA-NF-X (X ˆ CB, HF) group covalently bound to the CF3
moiety is far from 1208: this angle is actually close to 109.08
for each starting structure of the peptides studied.
In contrast, the structures
[a] Atom tags are defined in Figure 11.
120.88, which could suggest an sp2-type geometry. However,
À
the NF H bond does not lie in the same plane as the two
carbons bound to the NF atom, in agreement with the results
of X-ray diffraction on the PMR peptides 3a, 9a, 9c and 13.
Analysis of 15c, obtained by substitution of one of the
hydrogen atoms on the primary amino group of 15a with -
CH2CONH2, confirms the tendency of the substituted a-CF3
amino group NF to adopt a bond angle value around 1208, with
an overall local geometry similar to that of 15b, thus
confirming the highly distorted sp3 geometry already evi-
denced for the NF 15b.
Three analogues of 15b, namely 16 18 (see Figure 12),
characterized by the different number of F atoms in the
methyl group bound to the NH-substituted CA atom, were
studied next with the aim of gaining deeper insight into the
obtained after the quantum
mechanical level optimization
(see Figure 11 and data report-
ed in Table 5) clearly show that
when a substituent (X ˆ CB) is
present on the NH group a
widening of the CA-NF-CB angle
to a value around 1208 is ob-
served, matching the experi-
mentally determined X-ray
structure. This underlines the
importance of the correct theo-
retical treatment of this kind of
peptide models.
The NH2 group in 15a is
À
bound to a CA CF3 system with
no further ramification. This
primary amine nitrogen dis-
plays the typical structural char-
acteristics of an sp3 atom (Ta-
ble 5). Increasing the degree of
substitution on the amino group
by the insertion of a CH3 moi-
ety (15b), determines dramatic
effects on the bond angle on
nitrogen. In fact, the CA-NF-CB
bond angle becomes clearly
distorted. In the optimized
Figure 11. Optimized geometries for the structures for TI, and for the amides 15a c.
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org Chem. Eur. J. 2003, 9, 4510 4522
structure it adopts a value of
4518

Pseudopeptides
4510 4522
effect of increasing fluorine substitution on the geometry of
NH. Inspection of Table 5 shows that the CA-NF-CB angle
tends to assume a value closer to 1208 as the fluorine atom
number increases. This can be likely interpreted in terms of an
increasing steric hindrance of the fluoromethyl group cen-
tered on CF, which determines a progressive widening of the
CA-N-CB angle, which is particularly distorted in the case of
the bulky CF3 group. This hypothesis is supported by analysis
of the 203 crystal structures having secondary amine functions
found in the CCD (see Supporting Information). It is indeed
apparent that the largest C-NH-C bond angles are displayed
by those compounds having sterically demanding substituents
bound to the a-amino carbon. In order to completely rule out
the possibility that factors other than steric hindrance are
involved in the distortion of the CA-NF-CB angle, population
analysis was run on the optimized structure. The results
influencing the dipolar characteristics of each molecule. The
concentration of negative charge is located in a position
strictly correlated to the one of the negatively charged O
atoms in molecule TI. The presence of a net negative charge
on TI makes the overall MEP more negative. The absence of
such a charge in the fluorine containing peptidomimetics
favors the presence of positively or neutrally charged patches
on the MEP surface.
The presence of such a localized negatively charged region
can be extremely important, together with the peculiar
geometrical aspects of the NH groups of these compounds,
for the recognition of the designed peptides by putative
receptors.
À
showed that, as expected, the CA NF bond involving the
amino group flanking the CF3 substituent does not have any
double bond character.
Conclusion
In the case of TI the analogous angle (defined by the
tetrahedral carbon bearing two oxygen atoms, the NH group
and the CB attached to the N atom) displays a value of about
118.78. This value is very close to the value of the angle CA-NF-
CB calculated for the fluorinated peptidomimetics.
The stereoelectronic features of the three model CF3-
amides 15a c were evaluated with the calculation of their
molecular electrostatic potential (MEP); the surfaces of the
amides are displayed in Figure 12. All the molecules display a
high local concentration of negative charge on the CF3 group,
Conceptually new types of peptidomimetics, PMR and PMRI
y[NHCH(CF3)]Gly peptides, are now available through the
effective solution-phase chemistry described in this paper, as
well as through solid-phase chemistry, as previously repor-
ted.[7d] Several PMR y[NHCH(CF3)]Gly peptides investigat-
ed by X-ray diffraction show a remarkable proclivity to adopt
turn-like conformations independent of the presence of
ˆ
C O ¥¥¥ Hb-N intramolecular hydrogen bonding, which was
shown to be of paramount importance for stabilizing the turn-
like conformations adopted by Gellman×s unfluorinated
retropeptides. This is clearly shown by the strong similarity
in the values of the torsion angles defining the turns in the
crystal structure of compounds 3a, 9a, and 13, having the
same syn-configuration at the C13 and C15 stereocenters,
among which only 13 displays intramolecular hydrogen
bonding. In contrast, no turn-like structure is displayed by
9c, which has anti-configuration at the same centers. Similar
turn-like conformational states were found to be highly
populated in CDCl3 solution for y[NHCH(CF3)]-PMR tri-
peptides 12 (which is an exact structural analogue of one of
Gellman×s retropeptides), 13 and 14. These striking conforma-
tional features can be ascribed to the heavy stereoelectronic
demands of the CF3 group,[31] and possibly to its preferential
solvation once the turn-like conformation has been estab-
lished. In fact, it is apparent that in such turn-like conforma-
tions the CF3 group points towards the outside position of the
turn itself, independent of the other substituent groups
examined. These findings, which deserve further investiga-
tion, suggest that the CF3 group itself might constitute an
important stabilizing factor for the turn. This could represent
a new general concept for the rational design of linear
peptidomimetics incorporating a turn-like secondary struc-
ture. Finally, ab initio computational studies showed that the
[-CH(CF3)NH-] group has very peculiar characteristics, which
are intermediate between a peptide bond mimicand a
proteolytictransition state analogue. We believe that
y[NHCH(CF3)] PMR peptides hold the potential to become
an important class of peptidomimetics in biomedicinal and
pharmaceutical field.
Figure 12. The molecular electrostatic potential surfaces (MEP) for the
tetrahedral intermediate TI, and CF3-amides 15a c. The color code is from
blue for positive potential to red for negative potential.
Chem. Eur. J. 2003, 9, 4510 4522
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4519

A. Volonterio, M. Zanda et al.
FULL PAPER
with
points.
a
908 shifted sine-bell squared function to 1 k  1 k real data
Experimental Section
Molecular modelling on 13: Molecular models were build using a Silicon
Graphics 4D35GTworkstation running the Insight II & Discover software.
Molecular mechanics (MM) and molecular dynamics (MD) were carried
out using CVFF as force field. The starting geometry of the compounds
were generated using standard bond lengths and angles. The models were
then energy minimised without NOE constraints, using a optimised cycle
until a maximum energy derivative of 4.18  10À2 kJmolÀ1 äÀ1 was reached.
The subsequent unrestrained molecular dynamics calculation, performed
for 100 ps at 1000 K temperature and sampling the trajectory every 1 ps,
followed by minimisation, led to different structures.
General details: THF was freshly distilled from Na; diisopropylamine was
freshly distilled from CaH2. In all other cases, commercially available
reagent-grade solvents were employed without purification. All reactions
where an organicsolvent was employed were performed under nitrogen
atmosphere, after flame-drying of the glass apparatus. Melting points (m.p.)
are uncorrected and were obtained on a capillary apparatus. TLC were run
on silica gel 60 F254 Merck. Flash chromatographies (FC) were performed
1
with silica gel 60 (60 200 mm, Merck). H NMR spectra were run at 250,
400 or 500 MHz. Chemical shifts are expressed in ppm (d), using
tetramethylsilane (TMS) as internal standard for 1H and 13C nuclei (dH
and dC ˆ 0.00), while C6F6 was used as external standard (dF 162.90) for 19F.
X-ray structure analysis of 3a: Colorless lath of 0.45 Â 0.10 Â 0.05 mm size,
orthorhombic P212121, a ˆ 5.2797(4), b ˆ 13.3423(9), c ˆ 27.2759(18) ä,
Vˆ 1921.4(2) ä3, Z ˆ 4, 1calcd ˆ 1.391 gcmÀ3, 2qmax ˆ 618, diffractometer
Siemens Smart CCD, MoKa (l ˆ 0.71073 ä), w scan, Tˆ 293(2) K, 15758
reflections collected of which 5668 were independent (Rint ˆ 0.0643), direct
primary solution and refinement on F2,[32] 256 refined parameters, amino
hydrogen atom located in a difference Fourier synthesis and refined with
Experimental for the aza-Michael reaction: Synthesis of 3,4c is described
as an example. To a stirred solution of (E,S)-2a[9] (80 mg, 0.27 mmol) and l-
1b (131 mg, 0.54 mmol) in dry CH2Cl2 (3.4 mL) was added neat sym-
collidine (0.14 mL, 1.08 mmol). After 64 h at RT the solvent was removed
in vacuo, the crude dissolved in EtOAc and washed once with a 1n HCl.
The organiclayer was dried over anhydrous Na 2SO4, filtered, the solvent
removed in vacuo, and the crude purified by FC (hexane/diethyl ether
65:35) affording diastereoisomerically pure 3c (116 mg, 78.5%) and its
minor diastereoisomer 4c (20 mg, 13.5%). 3c: Rf ˆ 0.21 (hexane/EtOAc
80:20); [a]2D3 ˆ ‡28.38 (c ˆ 1.0, CHCl3); m.p. 102 1038C; FTIR (film):
nÄmax ˆ 3449, 1794, 1700, 1385, 1258, 1120 cmÀ1; 1H NMR (250 MHz, CDCl3):
d ˆ 7.38 7.20 (m, 10H), 5.13 (d, J ˆ 12.1 Hz, 1H), 5.06 (d, J ˆ 12.1 Hz, 1H),
4.70 (m, 1H), 4.24 (m, 1H), 4.16 (dd, J ˆ 8.8, 2.8 Hz, 1H), 3.62 (m, 1H), 3.42
(dd, J ˆ 15.3, 3.6 Hz, 1H), 3.36 (m, 2H), 3.15 (dd, J ˆ 15.3, 9.6 Hz, 1H), 2.77
(dd, J ˆ 13.7, 10.1 Hz, 1H), 2.01 (m, 1H), 1.88 (brs, 1H), 0.96 (d, J ˆ 6,7 Hz,
3H), 0.85 (d, J ˆ 6,7 Hz, 3H); 19F NMR (250 MHz, CDCl3): d ˆ À77.5 (d,
J ˆ 6.7 Hz); 13C (250 MHz, CDCl3): d ˆ 174.8, 169.3, 153.6, 135.6, 135.4,
129.4, 129.0, 128.6, 128.4, 128.2, 127.4, 126.0 (q, J ˆ 282.3 Hz), 66.8, 66.6,
66.5, 55.7 (q, J ˆ 29.3 Hz), 55.5, 37.9, 36.3, 31.8, 19.3, 17.5; MS (70 eV): m/z
À
restrained N H bond length, other hydrogen atoms riding, R1[I > 2s(I)] ˆ
0.0471, wR2 (all data) ˆ 0.1123, residual electron density 0.168
(À0.149) eäÀ3, absolute structure could not be determined.
X-ray structure analysis of 9a: Needle colorless crystal with dimension of
0.15  0.2  0.8 mm, orthorhombic P212121, a ˆ 9.106(5), b ˆ 15.611(5), c ˆ
16.770(5) ä, Vˆ 1383(1) ä3, Z ˆ 4, 1calcd ˆ 1.205 gcmÀ3, m ˆ 0.868 mmÀ1
,
Siemens P4 diffractometer with graphite monochromated CuKa radiation
(l ˆ 1.54179 ä), q/2q scan technique, room temperature, a total of 3055
reflections (2862 unique, Rint ˆ 0.016) collected up to 1368 in 2q. Structure
solved by direct methods[33] and refined on F2,[32] amino hydrogen freely
refined, other hydrogen atoms riding, R1 ˆ 0.0397 (Rw ˆ 0.100) for 2283
observed reflections [I ꢁ2s(I)] and 280 parameters refined, and R1 ˆ
0.0517 (Rw ˆ 0.131) for all data, residual electron density of 0.126 and
‡
À0.104 eäÀ3
.
(%): 507 (8) [M ‡H], 371 (90), 91 (100).
X-ray structure analysis of compound 9c: Needle colorless crystal with
dimension of 0.2  0.3  0.8 mm, hexagonal R3, a ˆ 35.624(5), c ˆ
5.210(5) ä, Vˆ 5726(6) ä3, Z ˆ 9, 1calcd ˆ 1.254 gcmÀ3, m ˆ 0.870mmÀ1, Sie-
mens P4 diffractometer with graphite monochromated CuKa radiation (l ˆ
1.54179 ä), q/2q scan technique, room temperature, a total of 2644
reflections (2435 unique, Rint ˆ 0.019) collected up to 1308 in 2q. Structure
solved by direct methods[33] and refined on F2,[32] amino hydrogen freely
refined, other hydrogen atoms riding, R1 ˆ 0.0493 (Rw ˆ 0.126) for 2122
observed reflections [I ꢁ 2s(I)] and 256 parameters refined, and R1 ˆ
0.0575 (Rw ˆ 0.134) for all data, residual electron density of 0.286 and
4c: Rf ˆ 0.17 (hexane/EtOAc80:20); 19F NMR (250 MHz, CDCl3): d ˆ
À77.1 (d, J ˆ 7.2 Hz).
Typical procedure synthesis of the PMR peptides: A 30% aqueous H2O2
solution (0.86 mL, 0.85 mmol) followed by solid LiOH (5 mg, 0.21 mmol)
was added to a cooled solution of 3c (97 mg, 0.21 mmol) in THF/H2O 4:1
(1.5 mL) at 08C and under nitrogen atmosphere. After 60 min the reaction
was quenched with saturated aqueous Na2SO3, warmed to rt, and extracted
three times with AcOEt, and the combined organic layers were dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. Purification by FC
(hexane/AcOEt 1:1 until complete recovery of the oxazolidin-2-one and
then MeOH) afforded the acid (50 mg, 68%). To a stirred mixture of this
acid (30 mg, 0.08 mmol) and l-1a (12 mg, 0.08 mmol) in dry DMF (1 mL)
was added, at 08C under nitrogen atmosphere, neat sym-collidine
(0.032 mL, 0.85 mmol), followed by solid HOAt (11 mg, 0.08 mmol) and
solid HATU (31 mg, 0.08 mmol). After 40 min the solution was quenched
with 1n HCl, warmed to RT, and extracted with AcOEt, and the combined
organiclayers dried over anhydrous Na 2SO4, filtered, and concentrated in
vacuo. Purification by FC (hexane/AcOEt 75:25) afforded 9a (35 mg,
quant). Rf ˆ 0.34 (hexane/EtOAc70:30); [ a]2D3 ˆ À9.28 (c ˆ 0.9, CHCl3);
À0.169 eäÀ3
.
Ab initio calculations: All ab initio calculations were carried out using the
Gaussian 98[34] molecular orbital package on an Origin 200 Silicon Graphics
workstation. The initial geometry of each model peptide was chosen to
mimic the situation in the experimentally determined crystal structure (see
Tables 4 and 5, and Supporting Information). Initial geometries were built
using the Macromodel6.5 package.[35] Three model peptides were chosen
with different substitutions on the NH (labeled NF in Figure 11) group
bound to the CF3 carrying carbon atom. Structure 15a is characterized by
the presence of a CF3-CH-NH2 group, structure 15b by the presence of a
CF3-CH-NH-CH3 while structure 15c carries a CF3-CH-NH-CH2-CO-NH2.
These groups were chosen to mimic different steric and electronic features
of each peptide. The geometries were optimized at DFT level[36] using the
B3LYP[37] functional and the 6-311‡‡G** basis. The DFT B3LYP level of
theory is known to give very accurate energy values, and the use of a basis
set like 6-311‡‡G** guarantees that possible polarization effects due to
the presence of the fluorine atoms are taken into account.[38] Previous
calculations on peptide models using this approach have proved to yield
results in terms of peptide geometry which are comparable to those
obtained using much higher levels of theory. Population analysis was run on
the minimized structures at the RHF 6-31G** level.
1
m.p. 70 718C; FTIR (KBr): nÄmax ˆ 3324, 1745, 1647, 1152 cmÀ1; H NMR
(250 MHz, CDCl3): d ˆ 7.42 7.32 (m, 5H), 7.10 (brd, J ˆ 6.2 Hz, 1H), 5.20
(d, J ˆ 12.0 Hz, 1H), 5.11 (d, J ˆ 12.0 Hz, 1H), 4.59 (m, 1H), 3.74 (s, 3H),
3.55 (m, 1H), 3.46 (d, J ˆ 5.0 Hz, 1H), 2.65 (dd, J ˆ 15.4, 3.1 Hz, 1H), 2.43
(dd, J ˆ 15.4, 9.3 Hz, 1H), 2.00 (m, 2H), 1.44 (d, J ˆ 6.9 Hz, 3H), 0.96 (d,
J ˆ 6.6 Hz, 3H), 0.88 (d, J ˆ 6.6 Hz, 3H); 19F NMR (250 MHz, CDCl3): d ˆ
À73.6 (d, J ˆ 6.9 Hz); 13C (250 MHz, CDCl3): d ˆ 174.4, 173.5, 168.2, 135.5,
128.6, 128.4, 126.0 (q, J ˆ 283.0 Hz), 66.9, 65.5, 56.0 (q, J ˆ 29.6 Hz), 52.4,
‡
48.3, 36.1, 31.9, 19.0, 18.0, 17.6; MS (70 eV): m/z (%): 433 (3) [M ‡H], 297
(100), 91 (100).
NMR studies on the secondary structure of y[NHCH(CF3)]-PMR
peptides: 1H NMR spectra were measured in CDCl3 with a 600 MHz
spectrometer. The concentrations of the samples were 3, 0.7 and 0.04mm
with temperatures ranging from 293 to 213 K. Chemical shifts are given in d
and referred to the CDCl3 used as the solvent. 2D ROESY spectra were
acquired in the phase sensitive TPPI mode, with 2 k  512 complex FIDs,
spectral width of 9615 Hz, recycling delay 1.6 s, 56 scans, at 213 and 293 K
with mixing times of 300 ms. The spectra were transformed and weighted
Acknowledgement
We thank the European Commission (IHP Network grant ™FLUOR
MMPI∫ HPRN-CT-2002-00181), MIUR (Cofin 2002, Project ™Peptidi
4520
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 4510 4522

Pseudopeptides
4510 4522
Sintetici Bioattivi∫) and CNR for financial support. Politecnico di Milano is
gratefully acknowledged for fellowships to A.V. and F.B.
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[6] For clarity, throughout the paper we refer to the title compounds
only as PMR and PMRI y[NHCH(CF3)]Gly peptides. However
y[NHCH(CF3)] and y[CH(CF3)NH] derivatives such as, HO-
AA1-[CH(CF3)CH(R)C(O)]-AA2-OH and HO-AA1-[C(O)CH(R)-
CH(CF3)]-AA2-OH, respectively, constitute two completely different
classes of compounds, not related by any symmetry operation, when
AA1 = AA2. It is convenient to distinguish between the two forms
when they are referred to a parent peptide such as H-AA1-AAR-AA2-
OH.
[7] Small libraries of PMR and PMRI y[NHCH(CF3)]Gly peptides, as
well as peptidyl hydroxamates having moderate inhibitory activity
against MMP-9 (gelatinase B), have been already prepared through
solid-phase synthesis: a) A. Volonterio, P. Bravo, N. Moussier, Zanda,
M. Tetrahedron Lett. 2000, 41, 6517 6521; b) A. Volonterio, P. Bravo,
M. Zanda, Tetrahedron Lett. 2001, 42, 3141 3144; c) A. Volonterio, S.
Bellosta, P. Bravo, M. Canavesi, E. Corradi, S. V. Meille, M. Monetti,
N. Moussier, M. Zanda, Eur. J. Org. Chem. 2002, 428 438; d) M. Sani,
P. Bravo, A. Volonterio, M. Zanda, Collect. Czech. Chem. Commun.
2002, 67, 1305 1319; for
a
recent paper on
a novel class of
retropeptides incorporating
a stereodefined 3,3,3-trifluoroalanine
¬
mimic: e) M. Sani, L. Bruche , G. Chiva, S. Fustero, J. Piera, A.
Volonterio, M. Zanda, Angew. Chem. 2003, 115, 2106 2109; Angew.
Chem. Int. Ed. 2003, 42, 2060 2063.
[8] See for example: L. R. Scolnick, A. M. Clements, J. Liao, L. Crenshaw,
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and mildness of this aza-Michael reaction are due to the significantly
greater reactivity of 2 with respect to other crotonates, which in turn is
due to the concomitant presence of the electron-withdrawing CF3
group and imide function. For other aza-Michael reactions with
4-substituted acceptors see: c) A. J. Burke, S. G. Davies, C. J. R.
Hedgecock, Synlett 1996, 621 622; d) J. d×Angelo, J. Maddaluno, J.
Am. Chem. Soc. 1986, 108, 8112 8114; e) G. Cardillo, E. Di Martino,
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¡
Tomasini, A. Trere, J. Org. Chem. 1993, 58, 5615 5619; g) S. W.
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Baldwin, J. Aube, Tetrahedron Lett. 1987, 28, 179 182; h) M. Hirama,
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¬
Soleilhac, B. P. Roques, M. C. Fournie-Zaluski, J. Med. Chem. 1988,
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[5] For a preliminary communication on the synthesis of PMR and PMRI
y[NHCH(CF3)]Gly peptides: a) A. Volonterio, P. Bravo, M. Zanda,
Org. Lett. 2000, 2, 1827 1830; a conceptually related peptide bond
surrogate y[CH(CN)NH] has been reported: b) S. Herrero, M. L.
¬
¬
¬
ƒ
Suarez-Gea, R. Gonzalez-Muniz, M. T. GarcÌa-Lopez, R. Herranz, S.
ƒ
Ballaz, A. Barber, A. Fortuno, J. Del RÌo, Bioorg. Med. Chem. Lett.
1997, 7, 855 860; for some recent examples of fluorine containing
peptidomimetics: c) G. S. Garrett, S. J. McPhail, K. Tornheim, P. E.
Correa, J. M. McIver, Bioorg. Med. Chem. Lett. 1999, 9, 301 306;
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Chem. Eur. J. 2003, 9, 4510 4522
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4521

A. Volonterio, M. Zanda et al.
FULL PAPER
2293 2304; t) J. d×Angelo, J. Maddaluno, J. Am. Chem. Soc. 1986, 108,
8112 8114; u) B. Mezrhab, F. Dumas, J. d×Angelo, C. Riche, J. Org.
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M. Sibi, Tetrahedron 2002, 58, 7991 8035.
[25] CCDC-204501 204503 (3a, 9a and 9c) contain the supplementary
crystallographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge CrystallographicData Centre, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: (‡44)1223-336-033; or e-mail: deposit@ccdc.cam.
ac.uk).
[26] a) F. H. Allen, O. Kennard, Chem. Des. Autom. News. Soc. 1993, 31
39; b) the results of the search for secondary amine structures R-NH-
R1 are elaborated in the Supporting Information. Among them, 29
structures (including 13) with a RR1CH-NH-CHR2R3, such as 3a, 9a,
and 9c, were found. The C-N-C angles of these structures span from
111.94 to 119.938, therefore the values for PMR y[NHCH(CF3)]Gly
peptides fall within this range, although they are quite large and
shifted toward the upper limit value; c) it is not surprising at all that
3a, 9a and 9c do not display CO ¥¥¥ NH intramolecular hydrogen
bonds, which in contrast are present both in Gellman×s retropeptides
and in 13. In fact, none of these PMR y[NHCH(CF3)]Gly peptides
incorporate a framework B, mimicof the Gellman×s retropeptide unit
C (see ref. [19]). This is likely due to the fact that amide and imide
carbonyls are much better hydrogen-bond acceptors than carbonyls
belonging to ester functions.
[11] Diastereomerically pure 3 were smoothly isolated by flash chroma-
tography on silica gel.
[12] The stereochemistry of 3a was assigned by X-ray diffraction (see the
Section on ™Secondary structure of PMR y[NHCH(CF3)]Gly pep-
tide∫), while the stereochemistry of the other major diastereomers
3b j was assigned on the basis of their spectral and chemical-physical
similarities with 3a. The stereochemistry of 5a, major diastereomer
derived from d-Ala-OMe 1a, was determined by chemical correlation
with 3a after cleavage of the oxazolidinone auxiliary.
[13] a) D. A. Evans, K. T. Chapman, J. Bisaha, J. Am. Chem. Soc. 1988, 110,
1238 1256; b) D. A. Evans, T. C. Britton, J. A. Ellman, R. L. Dorow,
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[16] Only proline derived pseudo-tripeptide 9d, obtained as a ca. 3:1
mixture of isomers at the peptide bond, and tetrapeptide 10 were
isolated as foams.
[17] It can be roughly estimated that the pKb of the [NHCH(CF3)] group
should be 5 units lower than that of the parent unfluorinated group
[NHCH(CH3)]: a) R. E. Banks, J. C. Tatlow, B. E. Smart, Organo-
fluorine Chemistry: Principles and Commercial Applications, Plenum
Press, New York, 1994, pp. 68 69; see also: b) N. Sewald, K. Burger,
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V. P. Kukhar, V. A. Soloshonok), Wiley, Chichester, 1995, pp. 139
220.
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[31] See ref. [17], pp. 537 538.
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[21] These compounds were obtained as described in ref. [7c].
[22] It is worth noting that the temperature dependence of the NHb
chemical shifts of epimers 13 and 14 seems to be convergent at higher
temperature, which is more than reasonable for two closely similar
structures.
[23] We did not find any experimental evidence for intramolecular
hydrogen bonds involving NHa, despite the fact that it could form a
six-membered ring A. One possible reason is that NHa is a worse
hydrogen-bond donor, since it is less acidic than NHb.
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[38] P. Hudaky, I. Jakly, A. G. Csaszar, A. Perczel, J. Comput. Chem. 2001,
22, 732 751.
Received: February 24, 2003 [F4881]
[24] Also, for 14 free MD calculations were performed and, after
minimization without constraints, many different structures were
obtained.
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